Microorganisms that extracellularly secrete lipids and methods of producing lipid and lipid particles encapsulating lipids using said microorganisms

ABSTRACT

There are provided microorganisms having a property of producing a lipid containing unsaturated fatty acids as constituent fatty acids and extracellularly secreting the produced lipid encapsulated in lipid particles, methods of screening said microorganisms, as well as methods of efficiently producing a fatty acid-containing lipid using said microorganisms. Furthermore, there are provided lipid particles encapsulating a lipid containing unsaturated fatty acids, and foods, cosmetics, and animal feeds comprising said lipid particles added thereto. Artificially treated microorganisms or microorganisms collected from nature are grown on a solid medium, and microbial strains that form lipid particles at the periphery of the colonies and/or microbial strains that, when cultured in a transparent liquid medium, make the culture liquid cloudy are selected. The microorganisms obtained are cultured, lipid-containing lipid particles secreted in the culture liquid, are separated from the culture liquid, and the lipid is separated and purified.

This application is a Divisional application of copending U.S.application Ser. No. 11/606,983, filed Dec. 1, 2006, which is aDivisional application of U.S. application Ser. No. 09/807,541, filedApr. 13, 2001 (now U.S. Pat. No. 7,157,254, issued Jan. 2, 2007), whichis the National Stage of International Application No. PCT/JP00/05425,filed Aug. 11, 2000, which claims priority to Japanese PatentApplication No. 11-229509, filed Aug. 13, 1999.

FIELD OF THE INVENTION

The present invention relates to microorganisms having a property ofextracellularly secreting a lipid containing unsaturated fatty acidsencapsulated in small particles, or to microorganisms having a propertyof extracellularly secreting a lipid containing unsaturated fatty acidsthat have 18 carbons and three or more double bonds or 20 or morecarbons and two or more double bonds, and more specifically tomicroorganisms having a property of extracellularly secreting a lipidcontaining unsaturated fatty acids encapsulated in small particles, saidmicroorganisms being obtained by artificially treating microorganismsthat have an ability of producing and intracellularly accumulating alipid containing unsaturated fatty acids, or to microorganisms having aproperty of extracellularly secreting a lipid containing unsaturatedfatty acids that have 18 carbons and three or more double bonds or 20 ormore carbons and two or more double bonds, said microorganisms beingobtained by artificially manipulating microorganisms that have anability of producing and intracellularly accumulating a lipid containingunsaturated fatty acids that have 18 carbons and three or more doublebonds or 20 or more carbons and two or more double bonds, and to methodsof efficiently producing a lipid containing unsaturated fatty acidsusing said microorganisms, as well as to methods of screening saidmicroorganisms, and furthermore to lipid particles encapsulating a lipidthat contains unsaturated fatty acids, and to foods, cosmetics, andanimal feeds comprising said lipid particles added thereto.

BACKGROUND ART

In recent years, various biological activities of highly unsaturatedfatty acids have attracted attention. For example, arachidonic acid isconsidered to be a precursor of prostaglandins, thromboxanes,prostacyclins, leucotrienes and the like that have biological activitiessuch as uterine contraction and relaxation, vasodilation, andhypotensive activity. These substances have been under intensiveresearch, together with docosahexaenoic acid, in recent years, aselements essential for the development of babies and infants. Variousfoods, cosmetics, and animal feeds to which a lipid containing highlyunsaturated fatty acids such as γ-linolenic acid, dihomo-γ-linolenicacid, and eicosapentaenoic acid in addition to arachidonic acid anddocosahexaenoic acid have been added are also attracting attention, andsome of the products to which highly unsaturated fatty acids has beenadded are commercially available.

Accordingly, methods of efficiently producing these highly unsaturatedfatty acids also are being studied intensively.

For example, methods of efficiently producing arachidonic acid,dihomo-γ-linolenic acid, γ-linolenic acid, or eicosapentaenoic acid byfermentation have been developed using microorganisms belonging to, forexample, the genus Mortierella, specifically the subgenus Mortierella,that are known to produce highly unsaturated fatty acids such asarachidonic acid, dihomo-γ-linolenic acid, γ-linolenic acid, andeicosapentaenoic acid (Japanese Unexamined Patent Publication (Kokai)No. 63-44891, Japanese Unexamined Patent Publication (Kokai) No.63-12290, Japanese Unexamined Patent Publication (Kokai) No. 63-14696,Japanese Unexamined Patent Publication (Kokai) No. 5-91887, and JapaneseUnexamined Patent Publication (Kokai) No. 63-14697). There is also knowna method of producing mead acid using a mutant strain in which Δ12desaturation activity is decreased or missing, said strain beingobtained by effecting mutation to microorganisms belonging to genusMortierella subgenus Mortierella (Japanese Unexamined Patent Publication(Kokai) No. 5-91888).

Thus, the production of a lipid containing highly unsaturated fattyacids using microorganisms that produce said fatty acids is increasinglybecoming a major source of highly unsaturated fatty acids. Thesemicroorganisms have a property of not only using highly unsaturatedfatty acids they produce as constituents of the cell membrane but alsoaccumulating highly unsaturated fatty acids as fat and oil(triglycerides) containing them as constituent fatty acids in the cell.By utilizing the fats and lipids accumulated in the cell, highproductivity of highly unsaturated fatty acids have come to be secured.

In this conventional production method, the amount of fat and oilobtained per culture was a product of the cell mass of the microorganismobtained by culturing and the amount of fat and oil produced per cell,and thereby how to increase the cell mass and the amount of fat and oilper cell was a challenge to be solved in order to attain enhancedproduction of fat and oil. Research has so far revealed that theselection of culture conditions permits increases in both of the aboveto a certain extent, but there are certain limits to each of them withincreases in the cell mass being limited by physical factors such as thevolume of the culture tank and increases in the amount of fat and oilper cell being limited by physiological factors of the microorganismused.

On the other hand, when it is desired to utilize a lipid produced by andaccumulated in the cell of the microorganism, it is necessary to collectthe cells after culturing, process the cells with a mill etc., todisrupt the cell membrane, and then to extract the lipid accumulated inthe cell.

If substances produced by a microorganism could be secreted outside ofthe cell instead of being accumulated in the cell, the physiologicalburdens placed on the microorganism by the substances produced can bealleviated and thus the microorganism can continue to produce theproducts; in the isolation and extraction of microbial products as well,extraction from the culture only is needed, which provides an advantagethat treatment becomes simplified and microorganisms can be continuouslyprocessed as they remain alive.

Based on the above, efforts to extracellularly secret a lipidaccumulated in the cell have recently been made by Sakuzo Fukui et al.(BIO INDUSTRY 12: 36-46 (1995)). Sakuzo Fukui et al. conducted researchon the production and secretion of lipids by microorganisms to developnovel biological fuel that can replace fossil fuel, and havesuccessfully bred genus Trichosporon yeast to convert sugar andn-alkanes to lipids and to secret them outside of the cell. They furtherdemonstrated that the constituent fatty acid species of extracellularlysecreted triglyceride (TG) are oleic acid, palmitic acid, linoleic acid,and stearic acid. However, it has a drawback that the TG that isdirectly secreted outside of the cell is incorporated into the cellagain and metabolized.

However, microorganisms that have an ability to produce lipidscontaining unsaturated fatty acids that have 18 carbons and three ormore double bonds or 20 or more carbons and two or more double bonds,and that have a property of extracellularly secreting the producedlipid, or microorganisms that have an ability of producing anunsaturated fatty acid-containing lipid, and that secrete the producedlipid encapsulated in small particles instead of directly secreting themat the time of extracellular secretion thereof, are not known.

In a study on γ-linolenic acid production by molds described in“Microbially produced fatty acids and their uses”, Osamu Suzuki,Fragrance Journal 1.989 (6), pp. 67-75, it is reported that a surfactantwas added to a culture medium of a microorganism of the genus Mucor toallow the leakage of some of lipids outside of the cell. However, itrelates to methods of artificially destroying the cell membrane to allowlipids accumulated in the cell to leak out of the cell, and does notutilize the ability of the cell per se to extracellularly secrete lipidsproduced in the cell.

Thus, there is a need to find microorganisms that have an ability ofproducing a lipid containing unsaturated fatty acids that have 18carbons and three or more double bonds or 20 or more carbons and two ormore double bonds, and have a property of extracellularly secreting theproduced lipids, or microorganism that have an ability of producing anunsaturated fatty acid-containing lipid, and that have a property ofextracellularly secreting the produced lipids encapsulated in smallparticles, and to develop methods of efficiently producing lipids thatcontain unsaturated fatty acids using said microorganisms.

Incidentally, in order to find microorganisms that have novel abilities,the development of an efficient screening method for microorganismshaving such abilities is a prerequisite. In the above breeding of genusTrichosporon yeast that converts sugar and n-alkanes into lipids andextracellularly secrete them, the following screening method wasadopted. Thus, yeast colonies that appeared on an agar plate medium (YPDmedium etc.) are UV-treated (15 watts at a distance of 30 cm for 15minutes) (this treatment is intended to suppress the dispersion ofcolony cells during layering treatment and not to induce mutation), theUV-treated colony plates are layered with a YPD soft agar mediumcontaining 10⁵ cells of a test strains, and are then cultured at 28° C.for 2 days. As the test strains, the A-1 strain and the ole-1 strainhaving auxotrophy for saturated fatty acids and unsaturated fatty acids,respectively, are used and those colonies giving a larger halo (themicro colony ring of the test strain) around them are selected aslipid-secreting strains. The selection of lipid-secreting strainsemploys two media: a soft agar medium containing or not containinglipase.

This method has a major drawback that although it can be applied toscreening of yeast for which layering is possible, it cannot be appliedto screening of microorganisms for which layering is impossible. Thereis additional disadvantage that the assay method are complicated.

Thus, in order to find microorganisms that have an ability of producinga lipid containing unsaturated fatty acids that have 18 carbons andthree or more double bonds or 20 or more carbons and two or more doublebonds, said microorganisms having a property of extracellularlysecreting the produced lipids, there is a need for the development of ascreening method that permits simple and efficient screening of saidmicroorganisms and that can be applied to various microorganisms, andthe development of a new screening method for finding microorganismsthat can secrete the produced lipids encapsulated in small particles atthe time of extracellular secretion of the produced lipids.

DISCLOSURE OF THE INVENTION

Thus, the present invention intends to provide microorganisms that havean ability of producing a lipid containing unsaturated fatty acids thathave 18 carbons and three or more double bonds or 20 or more carbons andtwo or more double bonds, and have a property of extracellularlysecreting the produced lipids, or said microorganisms that have anability of producing unsaturated fatty acids, secrete the producedlipids encapsulated in small particles, and methods of efficientlyproducing said lipids using said microorganisms, as well as methods ofscreening said microorganisms. The present invention also intends toprovide lipid particles encapsulating a lipid containing unsaturatedfatty acids, and to foods, cosmetics, and animal feeds comprising saidlipid particles added thereto.

After intensive research to attain the above objectives, the inventorsof the present invention have found that by artificially treatingmicroorganisms that have an ability of producing and intracellularlyaccumulating a lipid containing unsaturated fatty acids, it is possibleto create microorganisms that have an ability of extracellularlysecreting the produced lipids encapsulated in small particles.

In order to obtain microorganisms having the desired ability from thegroup of artificially treated microorganisms or microorganisms collectedfrom nature, the following simple screening method has been found thatcould be easily practiced by a person skilled in the art. Thus, as aprimary screening, artificially treated strains or strains collectedfrom nature are grown on solid media, and thereby those strainsproviding lipid particles around the colonies are selected. Then as asecondary screening, the strains selected in the primary screening arecultured under shaking in a transparent liquid medium (4% glucose, 1%yeast extract, pH 6.0) at 28° C. for 2 days. Microorganisms thataccumulate lipids in the cell do not make the medium cloudy duringculturing, whereas microorganisms that extracellularly secrete theproduced lipids make the medium cloudy, and therefore, microorganismshaving an ability of extracellularly secreting lipids can be easilyscreened by a mere visual confirmation of the degree of cloudiness ofthe culture liquid.

Then, it was found that, the efficient extracellular secretion of thelipid particles encapsulating said lipids utilizing the microorganismsobtained may be effected by culturing the microorganisms in a mediumhaving enhanced glucose concentration and/or enhanced pH.

It was also found that the extracellularly secreted lipid particles canbe easily separated from the culture liquid by centrifugation and thatcentrifugation and chromatography in addition to extraction with commonorganic solvents can be used to isolate lipids in the lipid particles.

Furthermore, the inventors have found that since the lipid particlesencapsulating the lipids separated from the culture liquid have aproperty of being easily dispersed in water or hydrophilic substancesand stably retain the lipids against oxidation, the lipid particles maybe added as they are to foods, cosmetics, or animal feeds thereby toobtain foods, cosmetics, or animal feeds containing unsaturated fattyacids that have novel features, and have completed the presentinvention.

Embodiment for Carrying Out the Invention

According to the present invention, unsaturated fatty acids that have 18carbons and three or more double bonds, or 20 or more carbons and two ormore double bonds refer to, for example, 5,8,11,14-eicosatetraenoic acid(arachidonic acid), 8,11,14-eicosatrienoic acid (dihomo-γ-linolenicacid), 6,9,12-octadecatrienoic acid (γ-linolenic acid),5,8,11,14,17-eicosapentaenoic acid, 8,11,14,17-eicosatetraenoic acid,6,9,12,15-octadecatetraenoic acid (stearidonic acid),9,12,15-octadecatrienoic acid (α-linolenic acid),4,7,10,13,16,19-docosahexaenoic acid (DHA), 8,11-eicosadienoic acid,5,8,11-eicosatrienoic acid (Mead acid), 7,10,13,16-docosatetraenoicacid, 4,7,10,13,16-docosapentaenoic acid, 7,10,13,16,19-docosapentaenoicacid, and the like.

The present invention provides microorganisms that have a property ofextracellularly secreting a lipid containing unsaturated fatty acidsthat have 18 carbons and three or more double bonds or 20 or morecarbons and two or more double bonds, or microorganisms that have aproperty of extracellularly secreting a lipid containing unsaturatedfatty acids encapsulated in small particles. More specifically, thepresent invention provides microorganisms that can extracellularlysecret a intracellularly produced lipid directly or by encapsulating itin small particles, said microorganisms being obtained by artificiallytreating microorganisms that have an ability of producing andintracellularly accumulating a lipid containing unsaturated fatty acidsthat have 18 carbons and three or more double bonds or 20 or morecarbons and two or more double bonds, or that have an ability ofproducing and intracellularly accumulating a lipid containingunsaturated fatty acids.

As used herein, microorganisms that have an ability of producing andintracellularly accumulating a lipid containing unsaturated fatty acidsinclude, for example, conventionally known microorganisms that have anability of producing γ-linolenic acid or microorganisms that have anability of producing arachidonic acid, microorganisms that have anability of producing DHA, microorganisms that have an ability ofproducing ω9 highly unsaturated fatty acids, and the like.Microorganisms that have an ability of producing arachidonic acidinclude microorganisms belonging to genus Mortierella, genusConidiobolus, genus Pythium, genus Phytophthora, genus Penicillium,genus Cladosporium, genus Mucor, genus Fusarium, genus Aspergillus,genus Rhodotorula, genus Entomophthora, genus Echinosporangium and genusSaprolegnia. As microorganisms belonging to genus Mortierella subgenusMortierella, there can be mentioned Mortierella elongata, Mortierellaexigua, Mortierella hygrophila, Mortierella alpina, Mortierellaschmuckeri, Mortierella minutissima, and the like. Specifically therecan be mentioned strains Mortierella elongata IFO8570, Mortierellaexigua IFO8571, Mortierella hygrophila IFO5941, Mortierella alpinaIFO8568, ATCC16266, ATCC32221, ATCC42430, CBS219.35, CBS224.37,CBS250.53, CBS343.66, CBS527.72, CBS529.72, CBS608.70, and CBS754.68,and the like.

These strains are all available without limitations from the Instituteof Fermentation (IFO) in Osaka, Japan, and American Type CultureCollection (ATCC) in the U.S.A., and Centrralbureau voorSchimmelcultures (CBS). It is also possible to use Mortierella elongataSAM0219 (FERM P-8703) (FERM BP-1239), a microbial strain isolated fromthe soil by the present inventor. Microbial strains belonging to thesetype cultures or those isolated from nature may be used as they are, andspontaneous mutants obtained by effecting growth and/or isolation onceor more may be used wherein the mutants have different properties thanthat of the original strains.

In accordance with the present invention, as microorganisms having theabove ability of producing unsaturated fatty acids, there can be usedthe microorganisms in which at least one of the Δ5 desaturationreaction, the Δ6 desaturation reaction, the Δ9 desaturation reaction,the Δ12 desaturation reaction, the ω3 desaturation reaction, and chainelongation reaction is enhanced, or reduced or missing may be obtainedby for example, mutation treatment or gene manipulation described below.

As artificial treatment effecting the above microorganisms, there can bementioned mutation, gene manipulation, cell fusion and the like.Mutation according to the present invention can be conducted byconventional mutation treatments: for example effecting irradiationtreatment (X ray, gamma ray, neutron beams), ultra violet irradiation,and high temperature treatment to induce mutation; and by suspendingmicroorganisms in a suitable buffer etc., to which a mutagen is addedfollowed by incubating for a given time, which is diluted appropriatelyand inoculated on an agar medium to obtain colonies of mutant strains.

As mutagens, there can be mentioned alkylating agents such as nitrogenmustard, methyl methane sulfonate (MMS), andN-methyl-N′-nitro-N-nitrosoguanidine (NTG), base analogs such as5-bromouracil, antibiotics such as mitomycin C, base synthesisinhibitors such as 6-mercaptopurine, dyes such as proflavine, certaincarcinogens such as 4-nitroquinoline-N-oxide, and other compounds suchas manganese chloride and formaldehyde. Microorganisms used may be livecells (mycelia) or spores.

In gene manipulation, conventional gene recombinant technology is used.

From microorganism groups subjected to the above artificial treatment ormicroorganism groups collected from nature according to conventionalmethods, strains of interest may be isolated based on the followingmethod. As a primary screening, after strains subjected to artificialtreatment or strains collected from nature are plated on a solid medium,the presence of lipid particles around colonies are used as an index toselect strains, and then as a secondary screening, the strains selectedin the primary screening are evaluated in a liquid medium as to whetherthey secrete a lipid outside of the cell. In an evaluation method, forexample, 4 ml of a transparent liquid medium (4% glucose, 1% yeastextract, pH 6.0) is dispensed in a test tube, which is sterilized at120° C. for 20 minutes, and then one platinum loopful of the strain thatwas selected in the primary screening is inoculated and incubated undershaking at 28° C. for 2 days.

Microorganisms like those belonging to genus Mortierella subgenusMortierella that produce triglyceride having an unsaturated fatty acidas constituent fatty acids in the cell but do not secrete it outside ofthe cell, even when cultured in the above liquid medium, do not make themedium cloudy, but when the lipid produced is secreted outside of thecell the medium becomes cloudy, so that the microorganisms thatextracellularly secrete the lipid can be easily confirmed. When thecloudiness does not change after being allowed to stand for some time, alipid has been secreted as lipid particles, whereas when the lipid hasbeen directly secreted, a lipid layer rises up to the surface of themedium, so that the two can be easily discriminated from each other. Ascomponents of the medium used in the secondary screening, any liquidmedium that can become transparent may be selected as appropriate, andany composition of the medium may be selected that is suitable for thegrowth of the microorganism to be evaluated.

From among the strains selected in this manner, it is preferred toselect strains that have an ability to grow and to produce lipids equalto or better than the that of the parent strain used for artificialtreatment. Although microorganisms of interest may be selected fromeither one of the above primary screening or the above secondaryscreening depending on the microorganism to be selected or the purposeof the study, combination of the two can assure better selection. Whenthe above two screening methods are combined, either the primaryscreening or the secondary screening may be conducted first.

As a strain obtained by the above method, there can be used Mortierellaalpina SAM2241 (FERM BP-7272) or SAM 2242 that was derived fromMortierella alpina IFO 8568 by the present inventors and thatextracellularly secretes an intracellularly produced lipid encapsulatedin small particles, but the strain to be used is not limited, andstrains that extracellularly secrete the lipid intracellularly producedmay be readily obtained by the above screening method, all of which canbe used.

Microorganisms of the present invention obtained by the above screeningmethod are microorganisms that can be converted to spheroplast orprotoplast, and microorganisms of the present invention can be used toobtain protoplast in which the cell wall has been completely removed orspheroplast in which part of the cell wall remains. This property cannotbe observed in the parent strain used for artificial treatment such asmutation treatment, and is probably due to the fact that the mutationtreatment changed the structure of the cell wall and made it fragile.

A microbial strain obtained as described above that, for example,strains that extracellularly secretes an intracellularly produced lipidencapsulated in small particles in the following method may be used toobtain lipid particles or the lipid. In order to culture microorganismsof the present invention, the spores, the mycelia, or the precultureobtained by culturing in advance are inoculated into a liquid medium andcultured.

In the case of liquid media, the carbon sources used include, but arenot limited to, any of glucose, fructose, xylose, saccharose, maltose,soluble starch, molasses, glycerol, mannitol and the like that arecommonly used. As nitrogen sources, in addition to natural nitrogensources such as peptone, yeast extract, malt extract, meat extract,casamino acid, corn steep liquor, soybean protein, defatted soybean, andcottonseed meal, organic nitrogen sources such as urea, and inorganicnitrogen sources such as sodium nitrate, ammonium nitrate, and ammoniumsulfate can be used.

When desired, inorganic salts such as phosphates, magnesium sulfate,iron sulfate, and copper sulfate, and vitamins can also be used as tracenutrients. The concentrations of these medium components are not limitedas long as they do not adversely affect microbial growth. Generally fromthe practical viewpoint, carbon sources are in the range of 0.1 to 40%by weight and preferably 1 to 25% by weight. Furthermore, the sequentialaddition of carbon sources and/or increasing initial concentrations ofcarbon sources can promote extracellular secretion of lipids. Nitrogensources may be in the range of 0.1 to 10% by weight and preferably 0.1to 6% by weight, and the nitrogen sources may be feeded in the middle ofculturing.

Though the optimum growth temperature as used herein may vary dependingon the microorganism used, it is 5 to 40° C., preferably 20 to 30° C.,or after culturing at 20 to 30° C. to grow the cell mass, culturing at 5to 20° C. may be continued to produce a lipid containing unsaturatedfatty acids. By means of such temperature control, the amount of highlyunsaturated fatty acids in the produced fatty acids can be enhanced. pHof the medium is 4 to 10, preferably 5 to 9, and aerated agitationculture, shaking culture, or continuous or stationary culture may beconducted using bioreactors. By using the initial pH of 5 to 9,preferably 6 to 9, and more preferably 7 to 9, extracellular secretionof lipids can be promoted. Culturing is usually conducted for 2 to 30days, preferably 5 to 20 days, and more preferably 5 to 15 days.

In accordance with the present invention, by adding precursors of thedesired unsaturated fatty acids to the medium and then culturing, theproduction of the desired unsaturated fatty acids, for example5,8,11,14-eicosatetraenoic acid (arachidonic acid),8,11,14-eicosatrienoic acid (dihomo-γ-linolenic acid),6,9,12-octadecatrienoic acid (γ-linolenic acid),5,8,11,14,17-eicosapentaenoic acid, 8,11,14,17-eicosatetraenoic acid,6,9,12,15-octadecatetraenoic acid (stearidonic acid),9,12,15-octadecatrienoic acid (α-linolenic acid),4,7,10,13,16,19-docosahexaenoic acid (DHA), 8,11-eicosadienoic acid, and5,8,11-eicosatrienoic acid (Mead acid) can be promoted.

Precursors that may be used include, but not limited to, hydrocarbonssuch as tetradecane, hexadecane, and octadecane, fatty acids such astetradecanoic acid, hexadecanoic acid, and octadecanoic acid or salts(e.g. sodium salts and potassium salts) or esters thereof, or fat andoil containing fatty acids (e.g. olive oil, coconut oil, palm oil,flaxseed oil, fish oil, and microbial oil) as constituent ingredients.

By adding fat and oil containing, as constituent ingredients unsaturatedfatty acids (for example, fish oil and microbial oil), or saidunsaturated fatty acids to the medium and culturing, the microorganismsof the present invention incorporate the added unsaturated fatty acidsor fat and oil into the cell and extracellularly secrete the lipidcontaining said unsaturated fatty acids as lipid particles. Therefore,even if unsaturated fatty acids that are not originally produced by themicroorganism are used, it is possible to produce lipid particlesencapsulating the lipid containing such unsaturated fatty acids asconstituent fatty acids.

The total amount of the added substrate containing the above precursoris 0.001 to 10% by weight and preferably 0.5 to 10% by weight relativeto the medium. These substrates may be added either before orimmediately after inoculating the production microorganism, or after thestart of culturing, or they may be added at both time points. Theaddition after the start of culturing may be once or more than once onan intermittent basis. Alternatively, they may be added continuously.Alternatively, these substrates may be used as the sole carbon sourcefor culturing.

By culturing the microorganisms of the present invention as describedabove, a lipid containing unsaturated fatty acids can be produced andaccumulated in large quantities in the cell, and said lipid is secreteddirectly or as lipid particles encapsulated in small particles. When aliquid medium is used, lipid particles or the lipid can be harvestedfrom the culture or the cultured liquid from which cultured cells havebeen removed as in the following manner.

After culturing is over, cultured cells are separated from the cultureusing conventional means for separating solids and liquids such ascentrifugation and filtration to obtain a medium (referred to as cultureliquid) in which lipid particles encapsulating lipids are dispersed.From the culture liquid, lipid particles encapsulating lipids may beisolated as one containing medium components by lyophilization, or lipidparticles containing no medium components may only be isolated byconventional centrifugation or column treatment. For example, acentrifuge (TOMYRL-101) or a swing rotor (TS-7) may be used forcentrifugation at a maximum centrifugal force of 1000×g or greater,preferably 1500×g or greater for about 10 minutes to separate lipidparticles.

The lipid particles obtained in the above methods are composed ofsugars, proteins, and lipids. Their composition excluding watercomprises 0 to 70%, preferably 20 to 60%, of sugars, 0 to 40%,preferably 10 to 30%, of proteins, and 20 to 100%, preferably 30 to 80%,of lipids. However, the ratio of sugars, proteins, and lipids may varydepending on the culture condition, and is not limited to the aboveratio. In fact, water is included in some cases.

The lipid particles of the present invention have an average diameter of0.2 to 10 μm, preferably 2 to 4 μm with the diameter of the largestlipid particle being 40 μm, preferably 10 μm. They can be separated bycentrifugation; for example, a centrifuge (TOMYRL-101) or a swing rotor(TS-7) may be used for centrifugation at a maximum centrifugal force of1000×g or greater, preferably 1500×g or greater, for about 10 minutes toseparate lipid particles. Furthermore, the lipid particles of thepresent invention may be easily dispersed in water or hydrophilicsubstances, and they have a property of stably retaining lipids againstoxidation.

Harvesting of a lipid in the lipid particles can be carried out eitherdirectly from the culture liquid before separating the lipid particlesor from the lipid particles separated from the culture liquid. When itis extracted from the lipid particles separated from the culture liquid,it is extracted in a manner similar to that conventionally performed toextract lipids from the cells. Thus, lipid particles are extracted withan organic solvent under a stream of nitrogen. As organic solvents,ether, hexane, methanol, ethanol, chloroform, dichloromethane, petroleumether and the like can be used, and satisfactory results can also beobtained by alternate extraction with methanol and petroleum ether, orby extraction with a single layer solvent of chloroform-methanol-water.When they are directly extracted from the culture liquid beforeseparation of the lipid particles, similar organic solvents may be used,however, in practice the use of a solvent that can be separated fromwater is preferred, and considering their application to foods, the useof hexane is preferred. Evaporation of organic solvents from the extractunder reduced pressure yields a lipid containing unsaturated fattyacids.

In the lipids obtained as above, unsaturated fatty acids are present inthe form bound to triglyceride, phosphatidylcholine,phosphatidylethanolamine, phosphatidylinositol and the like. The lipidsare composed of glycerides (triglycerides, diglycerides,monoglycerides), phospholipids, fatty acids, glycolipids, sterol estersand the like, and glycerides in the lipid are 50 to 100%, preferably 70to 100%, phospholipids are 0-50%, preferably 0 to 30%, fatty acids,glycolipids, and sterol esters combined are 0 to 30%, preferably 0 to15%. Triglycerides in the lipids are 50 to 100%, preferably 70 to 100%.

The lipid composition in a lipid containing unsaturated fatty acidsproduced using the microorganisms of the present invention derived frommicroorganisms belonging to genus Mortierella is 70 to 100% by weight ofneutral lipids, 0 to 30% by weight of polar lipids, and triglyceride, amain component of the neutral lipids, is 70 to 99% by weight in thelipids. The contents of unsaturated fatty acids may vary depending onthe microorganisms and culture conditions used, and the content ofarachidonic acid relative to the total fatty acids is not smaller than10% by weight, preferably 20 to 100% by weight, and more preferably 40to 100% by weight. The ratio of arachidonic acid to the total fattyacids in triglycerides is not smaller than 10% by weight, preferably 15to 100% by weight, and more preferably 35 to 90% by weight. However, thepresent invention is not limited to lipid particles secreted bymicroorganisms belonging to subgenus Mortierella, and fatty acidsproduced are not limited to arachidonic acid, either. Furthermore, afterallowing the cell to incorporate the lipid added to the culture medium,they may be allowed to be secreted as lipids in lipid particles, andthereby the lipid composition of lipids in lipid particles and the ratioof fatty acids of interest are varied, and in a sense can be freelydesigned.

In order to isolate and purify triglycerides containing unsaturatedfatty acids from an unsaturated fatty acid-containing lipid collectedfrom the lipid particles or the culture liquid, standard methods areused such as deoxygenation, degumming, dehydration, steam distillation,molecular distillation, cooling separation, and column chromatography.

In order to separate unsaturated fatty acids from a lipid containingunsaturated fatty acids, they are used in the form of mixed fatty acidsor mixed fatty acid esters and concentrated and separated byconventional methods such as urea addition, cooling separation, andcolumn chromatography.

The lipid particles of the present invention contain, in abundance,unsaturated fatty acids in the form of triglycerides. Their applicationsinclude, but not limited to, raw materials of foods, beverages,cosmetics, pharmaceuticals, animal feeds and the like and additivesthereof. Besides, their objective of uses and the amount used are notlimited in any way.

For example, foods include, in addition to general foods, functionalfoods, nutrient supplements, formula for premature infants, formula forbabies, baby foods, foods for pregnant women or foods for the agedpeople. The lipid particles of the present invention have excellentdispersion properties to water or hydrophilic substances, and thereforethey can be added to foods containing no fat and oil the addition towhich having conventionally been impossible, in particular variousbeverages. Furthermore, since the lipid particles of the presentinvention can stably retain lipids against air oxidation, the additionto foods containing conventional fat and oil can be effected moreeasily. As foods containing fat and oil, there can be mentioned, forexample, natural foods originally containing fat and oil such as meat,fish, or nuts, foods such as soup to which fat and oil are added duringcooking, processed foods that uses fat and oil as heat medium such asdoughnuts, fat and oil foods such as butter, processed foods to whichfat and oil are added during processing such as cookies, or foods towhich fat and oil are sprayed or pasted during processing finishing suchas hard biscuits. Furthermore, they may be in the form of functionalfoods and pharmaceuticals, and for example they may be in the processedform such as enteric nutrients, powders, granules, troches, medicines,suspensions, emulsions, syrups, and drinks.

EXAMPLES

The present invention will now be explained in more details withreference to specific examples. It should be noted, however, that thepresent invention will not limited by these examples in any way.

Example 1 Obtaining a Microbial Strain that Extracellularly SecretesLipid Particles by Mutation of Mortierella alpina IFO8568

Mortierella alpina IFO8568 was inoculated into a large slant bottlecontaining 300 ml of Czapek agar medium (0.2% NaNO₃, 0.1% K₂HPO₄, 0.05%MgSO₄.7H₂O, 0.05% KCl, 0.01% FeSO₄.7H₂O, 3% sucrose, 2% agar, pH 6.0),and was cultured at 28° C. for 2 weeks.

After culturing, 50 ml of sterile water to which had been added 2 dropsof Tween 80 was added to the large slant bottle, which was shakensufficiently, and then filtered with 4 ply gauze. This procedure wasrepeated twice, and the filtrate was centrifuged at 8000×g for 10minutes. Spores thus obtained were suspended into 50 mM Tris/maleatebuffer solution (pH 7.5) to 1×10⁶ /ml to prepare a spore solution.

To 1.0 ml of the spore solution thus obtained, 0.5 ml of 100 mMTris/maleate buffer solution (pH 7.5) was added, and 500 μl of the NTGsolution (5 mg of N-methyl-N′-nitro-N-nitrosoguanidine per ml ofdeionized water) was added, which was subjected to mutation treatment byincubating at 28° C. for 15 minutes. After adding 3 ml of 10% Na₂S₂O₃,the reaction mixture was centrifuged at 5500×g for 10 minutes, and theprecipitate (spores subjected to mutation treatment) was washed with 3ml of sterile water and centrifuged at 5500×g for 10 minutes, to whichsterile water was added to prepare a NTG-treated spore suspension.

The NTG-treated spore suspension was diluted to about 10⁻³ to 10⁻⁴ andthen plated on a GY agar plate (1% glucose, 0.5% yeast extract, 0.005%Triton X-100, 1.5% agar, pH 6.0). After incubating at 28° C., those thatdeveloped colonies were examined for morphology with a result thatmicrobial strains having a growth morphology distinctly different fromthat of the parent strain were obtained. The entire colonies of thehighly unsaturated fatty acid-producing microbial strains including theparent strain were covered with mycelia as the strain accumulate theproduced lipids in the cell, whereas the mutants obtained were coveredwith lipid particles.

Subsequently, 4 ml of a transparent liquid medium (4% glucose, 1% yeastextract, pH 6.0) was dispensed into a test tube and sterilized at 120°C. for 20 minutes. Then a platinum loopful of the microbial strainobtained above was inoculated thereinto and cultured under shaking at28° C. for 2 days, which made the medium cloudy. Even after allowing themedium to stand for over 10 minutes, no lipid layers were observed onthe surface of the medium. Thus, the lipid was possibly secreted aslipid particles.

The lipid particles that covered the colonies obtained in the culturingon the GY agar plate were analyzed for lipid by thin layerchromatography (TLC). To a previously activated plate (Merck 5554,200×200×0.25 mm, silica gel 60F-254, aluminium sheet), the sample andthe control (phospholipids, triglycerides, fatty acids) were plated,which was developed with n-hexane:diethylether:acetic acid=80:20:2(V/V/V) using phosphomolybdic acid (10% phosphomolybdic acid in ethanol)and primulin (0.01% primulin in 80% acetone) as a color developer. Forprimulin, bands were examined under UV light of a long wavelength (366nm). As a result, the majority of the lipids in the lipid particlesobserved outside of the cell were found to be triglycerides.

Thus, about 3000 colonies yielded mutants Mortierella alpina SAM2241FERM BP-7272 and SAM2242 that extracellularly secrete lipid particlesencapsulating mainly triglycerides having highly unsaturated fatty acidsas constituent fatty acids.

Example 2 Fatty Acid Analysis of an Extracellularly Secreted Lipid whenMortierella alpina SAM2241 that Extracellularly Secretes Lipid Particleswas Cultured on Various Media

Four ml each of medium A, B, C, D, E, and F was distributed in a testtube, and was sterilized at 120° C. for 20 minutes. A platinum loopfulof Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example 1 wasinoculated into the medium, and cultured under shaking at 28° C. for 2days and then at 12° C. for 7 days. After culturing, the cells and thefiltrate were separated by filtration. The filtrate obtained was placedin a screw-capped test tube (16.5 mmϕ), and lyophilized. To this wereadded 1 ml of methylene chloride and 2 ml of anhydrousmethanol-hydrochloric acid (10%), which was methylesterified by treatingat 50° C. for 3 hours. Four ml of n-hexane and 1 ml of water were addedto this, and then extracted twice. The solvent after extraction wasevaporated using a centrifuge evaporator (40° C., 1 hour), and the fattyacid methylesters thus obtained were analyzed by capillary gaschromatography. At the time of adding the methylester, 0.2 mg/mln-heptadecanoic acid (17:0) was added as an internal standard, and fattyacids were quantitated based on the ratio of surface area of GLC.

Medium A Glucose 1.0% K₂HPO₄ 0.3 MgSO₄•7H₂O 0.02 Polypeptone 1.5 NaCl0.2 Yeast extract 0.1 pH 7.0 Medium B Polypeptone 1.0% Meat extract 0.5Yeast extract 0.1 NaCl 0.5 pH 7.0 Medium C Glucose 5.0% Polypeptone 0.5KH₂PO₄ 0.2 K₂HPO₄ 0.1 MgSO₄•7H₂O 0.02 Yeast extract 0.1 pH 6.5 Medium DGlucose 2.0% Yeast extract 1.0 Bactopeptone 1.0 Medium E Glucose 2.0%(MRS medium) Meat extract 1.0 Yeast extract 0.5 Casein trypsin digest1.0 K₂HPO₄ 0.2 Sodium acetate 0.5 Diammonium citrate 0.2 MgSO₄•7H₂O 0.02MnSO₄•7H₂O 0.02 Tween 80 0.1 Medium F Glucose 1.0% Yeast extract 0.5 pH6.5

The result is shown in Table 1.

TABLE 1 The composition of extracellularly secreted fatty acids in thelipid obtained on various media 18:1 18:2 18:3 16:0 18:0 (n-9) (n-6)(n-6) DGLA AA Others Medium A 14 8 20 11 4 4 31 8 Medium B 15 6 28 16 9— 26 — Medium C 17 14 13 10 3 2 37 4 Medium D 14 9 23 9 5 2 31 7 MediumE 16 3 30 11 7 2 27 4 Medium F 15 5 23 10 5 3 32 716:0, palmitic acid; 18:0, stearic acid; 18:1 (n-9), oleic acid; 18:2(n-6), linoleic acid; 18:3 (n-6), γ-linolenic acid; DGLA,dihomo-γ-linolenic acid; AA, arachidonic acid

In any of the media, a lipid containing unsaturated fatty acids wereobserved to be extracellularly secreted. Furthermore, the total amountof the extracellularly secreted lipid obtained for medium A to F and theamount of arachidonic acid were found to be positively correlated withglucose concentration. Thus, the total amount of lipid per test tube was0.18 and 0.6 mg at a glucose concentration of 1%, 0.53 and 0.96 mg at aglucose concentration of 2%, and 2.11 mg at a glucose concentration of5%, and the amount of arachidonic acid per test tube was 0.05 and 0.15mg at a glucose concentration of 1%, 0.11 and 0.19 mg at a glucoseconcentration of 2%, and 0.62 mg at a glucose concentration of 5%.

A result with similar tendency was obtained for the mutant SAM2242.

Example 3 The Amount of Arachidonic Acid Produced by Aerated AgitatingCulture Using a 10 L Jar Fermentor of Mortierella alpina SAM2241 thatExtracellularly Secretes Lipid Particles

Five liters of a medium (A: pH 5.0, B: pH 6.0, C: pH 7.0) containing 2%glucose, 1.5% soy flour, 0.3% KH₂PO₄, 0.1% Na₂SO₄, 0.05% MgCl₂.6H₂O,0.05% CaCl₂.2H₂O, and 0.2% soybean oil was placed in a 10 L jarfermentor, and sterilized at 120° C. for 30 minutes. Mortierella alpinaSAM2241 (FERM BP-7272) obtained in Example 1 was inoculated therein, andwere subjected to aerated agitating culture at an aeration rate of 1.0vvm and a culture temperature of 24° C. for 10 days. 2.0% glucose wasadded on day 1 of culturing, 1.5% glucose on day 2, 1.0% glucose on days3 and 4, and 0.5% glucose on days 5 and 6.

Sampling was carried out every day. The culture liquid was separated byfiltration into the cells and the filtrate (extracellularly secretedlipid particles are dispersed therein). The cells were dried at 105° C.for 2 hours, and 20 mg of the dried cells was placed in a screw-cappedtest tube (16.5 mmϕ) and was subjected to methylesterification as inExample 2. The filtrate (1 ml) was placed in a screw-capped test tube(16.5 mmϕ), was lyophilized, and then was subjected tomethylesterification as in Example 2. The fatty acid esters thusobtained were analyzed by capillary gas chromatography. Table 2 showsthe amount produced of arachidonic acid and its content on day 9 ofculturing.

TABLE 2 The amount produced of arachidonic acid and its content on day 9of culturing Extracellular arachidonic acid Outside of the percentage Inthe cell cell (%) Medium A pH 5.0 6.4 g/L (33.2%) 0.14 g/L (37.4%) 2.1Medium B pH 6.0 5.8 g/L (32.4%) 0.24 g/L (34.5%) 4.0 Medium C pH 7.0 3.6g/L (29.7%) 0.43 g/L (30.8%) 10.7

Figures in parentheses indicate the ratio of arachidonic acid relativeto the total fatty acids.

With increased pH of the medium, the extracellular secretion ofarachidonic acid-containing lipids was promoted.

Example 4 Lipid Analysis of Lipid Particles Secreted by Mortierellaalpina SAM2241 that Extracellularly Secretes Lipid Particles

The culture filtrates on day 9 of culturing in medium A, B, and Cobtained in Example 3 were treated with chloroform/methanol/water(1:2:0.8) by the Blight-Dyer method and the total lipids were extractedfrom the extracellularly secreted lipid particles. The total lipidsobtained contained neutral lipids (triglycerides) and polar lipids(phospholipids). The total extracted lipids were charged into theSep-pak Silica cartridge (manufactured by Waters) and eluted to obtainthe neutral lipid fraction with chloroform and the polar lipid fractionwith methanol. After evaporating the solvent, methylesterification wascarried out as in Example 2, and fatty acid methylesters obtained wereanalyzed by capillary gas chromatography. As a percentage of the totalfatty acids to which triglycerides and phospholipids bind, thepercentages of triglyceride and phospholipids were calculated. As aresult, the percentage of the triglycerides in the total lipids formedium A, B, and C were 95.3%, 97.7%, and 96.2%, respectively.

Example 5 Continuous Culture of Mortierella alpina SAM2241 thatExtracellularly Secrets Lipid Particles in a 10 L Bioreactor

To a 10 L bioreactor having two built-in ceramic filters, 5 L of amedium containing 2% glucose and 2% yeast extract with pH adjusted to 7was prepared, to which a precultured microbial strain of Mortierellaalpina SAM2241 (FERM BP-7272) obtained in Example 1 was inoculated andsubjected to aerated agitating culture. On the next day, a glucosesolution was added through a ceramic filter to increase the glucoseconcentration in the medium by 3%. On day 2 also, the glucose solutionwas added through the ceramic filter to increase the glucoseconcentration in the medium by 3%.

On day 3 and after, a 5% glucose solution and a 0.05% yeast extractsolution were continuously passed through a ceramic filter at a speed ofabout 1000 ml/day. And the culture liquid was continuously extractedthrough a ceramic filter at about 600 ml/day (the amount of liquid isadjusted to remain constant). In order to prevent the clogging of thefilter with the cells, feeding of the glucose and yeast extractsolutions and extracting of the culture liquid were alternately carriedout as appropriate. Due to evaporation of water vapor by aeration, theamount of liquid in the jar remained almost constant. The feeding speedof glucose was adjusted by the glucose concentration to be extracted.

As a result, a medium (culture liquid) that contained about 1 g/Larachidonic acid-containing triglycerides was able to be continuouslyextracted.

Example 6 Microbial Transformation of the Fat and Oil Added to theMedium and its Migration into Lipid Particles by Mortierella alpinaSAM2241 that Extracellularly Secretes Lipid Particles

To 2 ml of a medium (pH 6.0) containing 1% glucose and 1% yeast extract,2% linseed oil or fish oil was added, which was then put into a 10 mlErlenmeyer flask and sterilized at 120° C. for 20 minutes. One platinumloopful of Mortierella alpina SAM2241 (FERM BP-7272) obtained in Example1 was inoculated into the medium, and cultured using a reciprocatingshaker (150 rpm) at 28° C. for 8 days. The filtrate was recovered byfiltration, was lyophilized, and then the extracellularly secreted lipidwas subjected to methylesterification as in Example 2, and the fattyacid methylester was analyzed by capillary gas chromatography.

When linseed oil was added to the medium, a major fatty acid of linseedoil, 9,12,15-octadecatrienoic acid (α-linolenic acid) served as asubstrate of the fatty acid biosynthetic enzymes of the mutant, and wasconverted to 6,9,12,15-octadecatetraenoic acid (stearidonic acid),8,11,14,17-eicosatetraenoic acid, and 5,8,11,14,17-eicosapentaenoicacid, and the lipid in the lipid particles contained 2.4, 3.3, and 8.1%of 6,9,12,15-octadecatetraenoic acid (stearic acid),8,11,14,17-eicosatetraenoic acid, and 5,8,11,14,17-eicosapentaenoicacid, respectively, confirming that the converted fatty acids areextracellularly secreted as the constituent fatty acids oftriglycerides. When fish oil was added to the medium, the fact that5,8,11,14,17-eicosapentaenoic acid and 4,7,10,13,16,19-docosahexaenoicacid of fish oil are incorporated into the microbial strain and areextracellularly secreted as constituent fatty acids of triglycerides wasconfirmed because the lipid of the extracellularly secreted lipidparticles contained 8.1 and 12.2% of 5,8,11,14,17-eicosapentaenoic acidand 4,7,10,13,16,19-docosahexaenoic acid, respectively.

Example 7 Component Analysis of Lipid Particles Extracellularly Secretedby Mortierella alpina SAM2241

In order to analyze components of extracellularly secreted lipidparticles, a spore suspension of Mortierella alpina SAM2241 obtained inExample 1 was plated to the GY agar plate (1% glucose, 0.5% yeastextract, 0.005% Triton X-100, 1.5% agar, pH 6.0), and cultured at 28° C.for 4 days. As shown in Example 1, the entire colonies were covered withlipid particles. Thus, the small particles were collected into ascrew-capped test tube (16.5 mmϕ). Chloroform (2 ml) and KCl solution (2ml) were added thereto, shaken and extracted. Lipids migrated into thechloroform layer and sugars and proteins migrated into the KCl layer,and these were analyzed for the components according to a standardmethod and were found to comprise sugars: 38.1%, proteins: 18.2%,lipids: 43.7%.

Example 8 Preparation of Formula Using Lipid Particles

The culture filtrate obtained in Example 3 was separated using acentrifuge (TOMY RL-101) at 1500×g and washed with sterile water toprepare lipid particles fit for consumption. The lipid particles (0.92g) were added to 100 g of powdered milk to prepare a formula containinglipid particles. The composition of arachidonic acid in the formulaobtained was 0.5% of the total fatty acids, which was similar to that ofthe mother's milk.

When the formula was dissolved in water, its dispersion in water wasgood and uniformly dispersed without any separation of oils.

Example 9 Preparation of capsules

Water was added to 100 parts per weight of gelatin and 35 parts perweight of food additive glycerin, which was dissolved at 50 to 60° C. toprepare a gelatin coating with a viscosity of 20000 cps. Then, from thelipid particles separated by centrifugation from the culture filtrateobtained in Example 3, lipids were extracted and purified according to astandard method. Then, 97% the refinded oil and 3% vitamin E oil weremixed to prepare a content. Using these, capsule molding and drying werecarried out according to a standard method so that soft capsulescontaining 180 mg content per capsule were produced.

Example 10 Preparation of Lipid Particles-Containing Beverages

The lipid particles (10 g) fit for consumption obtained in the methodshown in Example 8 were added to 10 L of orange juice to prepare juicecontaining lipid particles.

Reference to the microorganisms deposited under the Patent CooperationTreaty, Rule 13-2, and the name of the Depository Authority

Depository Authority:

Name: the National Institute of Bioscience and Human Technology, Agencyof Industrial Science and Technology Address: 1-3, Higashi 1-chome,Tsukuba city, Ibaraki Pref., Japan

Organism (1)

Name: Mortierella elongata SAM0219

Accession number: FERM BP-1239

Date deposited: Mar. 19, 1986

Organism (2)

Name: Mortierella alpina SAM2241

Accession number: FERM BP-7272

Date deposited: Aug. 11, 2000

The invention claimed is:
 1. A lipid composition comprising: a stearicacid content of 13 to 30% by weight over total fatty acids; an oleicacid content of 9 to 16% by weight over total fatty acids; a linoleicacid content of 3 to 9% by weight over total fatty acids; adihomo-γ-linolenic acid content of 0 to 4% by weight over total fattyacids; and an arachidonic acid content of 26 to 37% by weight over totalfatty acids.
 2. A food, a cosmetic, a pharmaceutical, or an animal feedcomprising the lipid composition of claim
 1. 3. The lipid composition ofclaim 1, wherein the lipid composition comprises: a stearic acid contentof 20 to 28% by weight over total fatty acids; an oleic acid content of10 to 11% by weight over total fatty acids; a linoleic acid content of 4to 7% by weight over total fatty acids; a dihomo-γ-linolenic acidcontent of 2 to 3% by weight over total fatty acids; and an arachidonicacid content of 27 to 32% by weight over total fatty acids.